Water Colors in Microbiology - Lab day 3

Day 3 of MMBL (a hip and savvy anachronism for Medical Microbiology Lab)

Today, we performed a few bacterial stains.  We kicked it off with a good old fashioned gram stain, to determine if our unknown bacteria was gram positive or gram negative (or gram variable).  On a fixed smear of bacteria, we poured crystal violet stain for about 20 seconds, then rinsed it off.  Next, we used a decolorizing agent (95% ethanol) to rinse the color off.  Finally, we covered it with safranin stain for about a minute before rinsing that off as well, and drying the slide with bibulous paper.  

If our sample was gram positive, it would have the crystal violet stain (a bluish purple), but if if was gram negative, it would have the safranin stain (a bright red).  This is because gram positive bacteria absorb the first stain, and it is not removed by the decolorizing agent.  Thus, the end result is that it keeps the first stain.  With gram negative bacteria, however, you can easily wash the first stain out with the decolorizing agent, allowing it to absorb the second stain (in this case, the safranin).  

Examining ours under a microscope, we determined that our sample was gram negative, because it had absorbed the safranin stain.

The next stain we did was a capsule stain.  We prepared a smear of bacteria in nigrosin stain (black), making sure we spread it over most of the slide.  After that, we again added the safranin stain, then rinsed it off.  We looked at it under a microscope, searching for capsules, which should be left unstained.  The nigrosin stains the bacteria, then the safranin stains the background.  Capsules don’t take any stain, so they should be left standing out.  Unfortunately, we were not able to see very clear capsules, but apparently this is not unusual.

We also did an endospore stain.  The point of this stain is to see if you have any endospores.  Over boiling water, we stained our bacterial smear with malachite green.  If there are endospores, then the green will stain them (so long as there’s heat).  After that, we rinsed off any excess stain, then again added safranin stain for about 90 seconds, before rinsing that off as well.  Using the oil immersion lens on the microscope, we looked for endospores.  We did not see any.  If they had been there, then they would be stained with malachite green, but all we observed was the safranin stain.

The last stain we did was an acid-fast stain, to determine if our bacteria was acid-fast, or non acid-fast based on the lipid content in their cell walls.  We used the Ziehl-Neelsen method (over hot water).  First, we put a paper over the slide, and saturated it with Ziehl-Neelson carbolfuchsin stain for about 3-5 minutes.  Next we used a decolorizing agent (acid-alcohol) to remove the color.  Finally, we coverd the smear with methylene blue for about 2 minutes, then rinsed off the excess.  We did this for two samples: our unknown, and another that Dr. Pathakamuri gave us.  Examining them under the microscope, we discovered that ours was not acid-fast, while the new sample was.